The development of PCR-based markers for molecular sex identification in the model insect species Tribolium castaneum

The red flour beetle, Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae), is a major pest of stored grain and cereal crops. It is also an important model species in genetic, ecological, and evolutionary research. The majority of its genome was recently sequenced and published. However, the genomic sequence of the small Y-chromosome is still undetermined, which hinders the development of molecular sex identification methods. Traditional methods for sexing adult forms of Tribolium beetles are troublesome. Therefore, a method for molecular sex identification in the red flour beetle was developed. One sex-linked randomly amplified polymorphic DNA marker was converted into a sequence-characterized amplified region (SCAR). The SCAR was aligned with the T. castaneum reference whole-genome sequence and fully matched a fragment of a single contig of unknown genomic location. The novelty of the method is that the fragment consists of shorter DNA fragments that are also present at other locations around the genome, but the particular order of these fragments within the sequenced region appeared to be Y-specific and this property was utilized for marker development. A set of three primers for multiplex PCR reaction was designed resulting in amplification of different length Y-specific and not-Y-specific (control) DNA fragments in a single PCR, which allows to distinguish males from females. The primers successfully sexed pre-sexed pupae and adult beetles from six laboratory strains, showing that the order of the repeated fragments is conserved in the species and is not strain-specific.     

Crinkled employs wingless pathway for wing development in Tribolium castaneum

Crinkled is associated with embryonic denticle formation and auditory organ development in Drosophila melanogaster. However, the functions of Crinkled have not been fully investigated. Additionally, the genes that participate in the Crinkled pathway are unknown. Phylogenetic analysis indicates that crinkled exhibits a one‐to‐one orthologous relationship in insects. In Tribolium castaneum, the crinkled gene is 6,498 bp in length and consists of six exons. Crinkled expression peaked during two phases in Tribolium: late embryonic and pupal stages. High levels of crinkled mRNA were detected in the fat body, head, epidermis, ovary, and accessory gland of late adults. Knockdown of crinkled using RNA interference (RNAi) severely affected wing morphogenesis in T. castaneum. We further showed that crinkled silencing reduced forked expression through wingless and shaven‐baby, and RNAi of forked phenocopied the effects of crinkled knockdown in T. castaneum. This study investigated the development role of crinkled in postembryonic stages and indicated that forked mediates the functions of crinkled during wing morphogenesis in T. castaneum.     

The role of Broad in the development of Tribolium castaneum: implications for the evolution of the holometabolous insect pupa

The evolution of complete metamorphosis in insects is a key innovation that has led to the successful diversification of holometabolous insects, yet the origin of the pupa remains an enigma. Here, we analyzed the expression of the pupal specifier gene broad (br), and the effect on br of isoform-specific, double-stranded RNA-mediated silencing, in a basal holometabolous insect, the beetle Tribolium castaneum. All five isoforms are weakly expressed during the penultimate instar and highly expressed during the prepupal period of the final instar. Application of hydroprene, a juvenile hormone analog, during the penultimate instar caused a repeat of the penultimate br expression patterns, and the formation of supernumerary larvae. Use of dsRNA against the br core region, or against a pair of either the br-Z2 or br-Z3 isoform with the br-Z1 or br-Z4 isoform, produced mobile animals with well-differentiated adult-like appendages, but which retained larval-like urogomphi and epidermis. Disruption of either the br-Z2 or the br-Z3 isoform caused the formation of shorter wings. Disruption of both br-Z1 and br-Z4 caused the appearance of pupal traits in the adults, but disruption of br-Z5 had no morphological effect. Our findings show that the br isoform functions are broadly conserved within the Holometabola and suggest that evolution of br isoform expression may have played an important role in the evolution of the pupa in holometabolous insects.     

Influence of nutrition on male development and reproduction in Tribolium castaneum

Tribolium beetles have evolved over several thousand years to colonize and exploit various food products that vary widely in their nutritional quality. Here Tribolium castaneum was used as a model organism to explore the effect of nutritional quality on male development and reproduction. The results showed, when tested across different qualities of nutritional diets, Tribolium males developed faster and their body size was larger on a high-quality diet, and there were significant correlations between male developmental traits. However, Tribolium males fed different nutritional diets did not show significant variation in olfactory attractiveness, mating rate, insemination rate, sperm defense (P1), sperm offense (P2), and reproductive success within a population context (RSPC). Moreover, there was no significant correlation of male reproductive performances except RSPC with developmental traits, and except for P2 and RSPC, no significant correlations between male reproductive performances. Therefore, although male developmental performance was significantly influenced by diet quality, reproductive performance was not. We discussed these findings and their sexual selection implications in light of its habitat.     

Palatogenesis: morphogenetic and molecular mechanisms of secondary palate development

Mammalian palatogenesis is a highly regulated morphogenetic process during which the embryonic primary and secondary palatal shelves develop as outgrowths from the medial nasal and maxillary prominences, respectively, remodel and fuse to form the intact roof of the oral cavity. The complexity of control of palatogenesis is reflected by the common occurrence of cleft palate in humans. Although the embryology of the palate has long been studied, the past decade has brought substantial new knowledge of the genetic control of secondary palate development. Here, we review major advances in the understanding of the morphogenetic and molecular mechanisms controlling palatal shelf growth, elevation, adhesion and fusion, and palatal bone formation.     

Morphogenesis and molecular mechanisms involved in human kidney development

The development of the human kidney is a complex process that requires interactions between epithelial and mesenchymal cells, eventually leading to the coordinated growth and differentiation of multiple highly specialized stromal, vascular, and epithelial cell types. The application of molecular biology and immunocytochemistry to the study of cell types involved in renal morphogenesis is leading to a better understanding of nephrogenesis, which requires a fine balance of many factors that can be disturbed by various prenatal events in humans. The aim of this paper is to review human kidney organogenesis, with particular emphasis on the sequence of morphological events, on the immunohistochemical peculiarities of nephron progenitor populations and on the molecular pathways regulating the process of mesenchymal to epithelial transition. Kidney development can be subdivided into five steps: (i) the primary ureteric bud (UB); (ii) the cap mesenchyme; (iii) the mesenchymal-epithelial transition; (iv) glomerulogenesis and tubulogenesis; (v) the interstitial cells. Complex correlations between morphological and molecular events from the origin of the UB and its branching to the metanephric mesenchyme, ending with the maturation of nephrons, have been reported in different animals, including mammals. Marked differences, observed among different species in the origin and the duration of nephrogenesis, suggest that morphological and molecular events may be different in different animal species and mammals. Further studies must be carried out in humans to verify at the morphological, immunohistochemical, and molecular levels if the outcome in humans parallels that previously described in other species.     

Individual and group selection for productivity in Tribolium castaneum

Summary The usefulness of a non-random group selection method in the improvement of a character influenced by interactions among individuals has been experimentally tested in a population of Tribolium castaneum. The selected trait —number of adults produced in a fixed period of time —showed a clear increase after 11 generations of selection. This increase is related to a reduction in developmental time, and it was specific for the population structure in which selection was applied, vanishing when this structure was altered.     

Tribolium castaneum Transformer-2 regulates sex determination and development in both males and females

Tribolium castaneum Transformer (TcTra) is essential for female sex determination and maintenance through the regulation of sex-specific splicing of doublesex (dsx) pre-mRNA. In females, TcTra also regulates the sex-specific splicing of its own pre-mRNA to ensure continuous production of functional Tra protein. Transformer protein is absent in males and hence dsx pre-mRNA is spliced in a default mode. The mechanisms by which males inhibit the production of functional Tra protein are not known. Here, we report on functional characterization of transformer-2 (tra-2) gene (an ortholog of Drosophila transformer-2) in T. castaneum. RNA interference-mediated knockdown in the expression of gene coding for tra-2 in female pupae or adults resulted in the production of male-specific isoform of dsx and both female and male isoforms of tra suggesting that Tra-2 is essential for the female-specific splicing of tra and dsx pre-mRNAs. Interestingly, knockdown of tra-2 in males did not affect the splicing of dsx but resulted in the production of both female and male isoforms of tra suggesting that Tra-2 suppresses female-specific splicing of tra pre-mRNA in males. This dual regulation of sex-specific splicing of tra pre-mRNA ensures a tight regulation of sex determination and maintenance. These data suggest a critical role for Tra-2 in suppression of female sex determination cascade in males. In addition, RNAi studies showed that Tra-2 is also required for successful embryonic and larval development in both sexes.     

Heavy meromyosin binding to microfilaments involved in cell and morphogenetic movements

Certain microfilaments found in migratory single cells and in morphogenetically-active epithelial cells are shown to bind heavy meromyosin prepared from skeletal muscle. This ‘actin-like’ feature of the filaments is compatible with the hypothesis that such filaments are components of contractile systems in these cells.     

Genomic and proteomic studies on the effects of the insect growth regulator diflubenzuron in the model beetle species Tribolium castaneum

Several benzoylphenyl urea-derived insecticides such as diflubenzuron (DFB, Dimilin) are in wide use to control various insect pests. Although this class of compounds is known to disrupt molting and to affect chitin content, their precise mode of action is still not understood. To gain a broader insight into the mechanism underlying the insecticidal effects of benzoylphenyl urea compounds, we conducted a comprehensive study with the model beetle species and stored product pest Tribolium castaneum (red flour beetle) utilizing genomic and proteomic approaches. DFB was added to a wheat flour-based diet at various concentrations and fed to larvae and adults. We observed abortive molting, hatching defects and reduced chitin amounts in the larval cuticle, the peritrophic matrix and eggs. Electron microscopic examination of the larval cuticle revealed major structural changes and a loss of lamellate structure of the procuticle. We used a genomic tiling array for determining relative expression levels of about 11,000 genes predicted by the GLEAN algorithm. About 6% of all predicted genes were more than 2-fold up- or down-regulated in response to DFB treatment. Genes encoding enzymes involved in chitin metabolism were unexpectedly unaffected, but many genes encoding cuticle proteins were affected. In addition, several genes presumably involved in detoxification pathways were up-regulated. Comparative 2D gel electrophoresis of proteins extracted from the midgut revealed 388 protein spots, of which 7% were significantly affected in their levels by DFB treatment as determined by laser densitometry. Mass spectrometric identification revealed that UDP-N-acetylglucosamine pyrophosphorylase and glutathione synthetase were up-regulated. In summary, the red flour beetle turned out to be a good model organism for investigating the global effects of bioactive materials such as insect growth regulators and other insecticides. The results of this study recapitulate all of the different DFB-induced symptoms in a single model insect, which have been previously found in several different insect species, and further illustrate that DFB treatment causes a wide range of effects at the molecular level.